Almost there: transmission routes of bacterial symbionts between trophic levels.
ABSTRACT Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts--Rickettsia and Hamiltonella--from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed.
- SourceAvailable from: Luiz Orlando de Oliveira[Show abstract] [Hide abstract]
ABSTRACT: Individual traits vary among and within populations, and the co-occurrence of different endosymbiont species within a host may take place under varying endosymbiont loads in each individual host. This makes the recognition of the potential impact of such endosymbiont associations in insect species difficult, particularly in insect pest species. The maize weevil, Sitophilus zeamais Motsch. (Coleoptera: Curculionidae), a key pest species of stored cereal grains, exhibits associations with two endosymbiotic bacteria: the obligatory endosymbiont SZPE (''Sitophilus zeamais Primary Endosymbiont'') and the facultative endosymbiont Wolbachia. The impact of the lack of SZPE in maize weevil physiology is the impairment of nutrient acquisition and energy metabolism, while Wolbachia is an important factor in reproductive incompatibility. However, the role of endosymbiont load and co-occurrence in insect behavior, grain consumption, body mass and subsequent reproductive factors has not yet been explored. Here we report on the impacts of co-occurrence and varying endosymbiont loads achieved via thermal treatment and antibiotic provision via ingested water in the maize weevil. SZPE exhibited strong effects on respiration rate, grain consumption and weevil body mass, with observed effects on weevil behavior, particularly flight activity, and potential consequences for the management of this pest species. Wolbachia directly favored weevil fertility and exhibited only mild indirect effects, usually enhancing the SZPE effect. SZPE suppression delayed weevil emergence, which reduced the insect population growth rate, and the thermal inactivation of both symbionts prevented insect reproduction. Such findings are likely important for strain divergences reported in the maize weevil and their control, aspects still deserving future attention. Citation: Carvalho GA, Vieira JL, Haro MM, Corrêa AS, Ribon AOB, et al. (2014) Pleiotropic Impact of Endosymbiont Load and Co-Occurrence in the Maize Weevil Sitophilus zeamais. PLoS ONE 9(10): e111396. doi:10.1371/journal.pone.0111396 Copyright: ß 2014 Carvalho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The financial support was provided by the National Council of Scientific and Technological Development (CNPq, Brazilian Ministry of Science and Technology), CAPES Foundation (Brazilian Ministry of Education), and Minas Gerais State Foundation of Research Aid (FAPEMIG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests. Prof. Raul Narciso C. Guedes is currently an academic editor of PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed in the online guide for authors.PLoS ONE 10/2014; 9(10). · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae) is an omnivorous insect used for biological control. Augmentative release and conservation of N. tenuis have been used for pest control in tomato crops. Intracellular bacterial symbionts of arthropods are common in nature and have diverse effects on their hosts; in some cases they can dramatically affect biological control. Fingerprinting methods showed that the symbiotic complex associated with N. tenuis includes Wolbachia and Rickettsia. Rickettsia of N. tenuis was further characterized by sequencing the 16S rRNA and gltA bacterial genes, measuring its amount in different developmental stages of the insect by real-time polymerase chain reaction, and localizing the bacteria in the insect's body by fluorescence in situ hybridization. The Rickettsia in N. tenuis exhibited 99 and 96% similarity of both sequenced genes to Rickettsia bellii and Rickettsia reported from Bemisia tabaci, respectively. The highest amount of Rickettsia was measured in the 5th instar and adult, and the symbionts could be detected in the host gut and ovaries. Although the role played by Rickettsia in the biology of N. tenuis is currently unknown, their high amount in the adults and localization in the gut suggest that they may have a nutritional role in this insect.Bulletin of entomological research. 07/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: Whiteflies (Hemiptera: Aleyrodidae) are sap-sucking insect pests, and some cause serious damage in agricultural crops by direct feeding and by transmitting plant viruses. Whiteflies maintain close associations with bacterial endosymbionts that can significantly influence their biology. All whitefly species harbor a primary endosymbiont, and a diverse array of secondary endosymbionts. In this study, we surveyed 34 whitefly populations collected from the states of Sao Paulo, Bahia, Minas Gerais and Parana in Brazil, for species identification and for infection with secondary endosymbionts. Sequencing the mitochondrial Cytochrome Oxidase I gene revealed the existence of five whitefly species: The sweetpotato whitefly Bemisia tabaci B biotype (recently termed Middle East-Asia Minor 1 or MEAM1), the greenhouse whitefly Trialeurodes vaporariorum, B. tabaci A biotype (recently termed New World 2 or NW2) collected only from Euphorbia, the Acacia whitefly Tetraleurodes acaciae and Bemisia tuberculata both were detected only on cassava. Sequencing rRNA genes showed that Hamiltonella and Rickettsia were highly prevalent in all MEAM1 populations, while Cardinium was close to fixation in only three populations. Surprisingly, some MEAM1 individuals and one NW2 population were infected with Fritschea. Arsenopnohus was the only endosymbiont detected in T. vaporariorum. In T. acaciae and B. tuberculata populations collected from cassava, Wolbachia was fixed in B. tuberculata and was highly prevalent in T. acaciae. Interestingly, while B. tuberculata was additionally infected with Arsenophonus, T. acaciae was infected with Cardinium and Fritschea. Fluorescence in situ hybridization analysis on representative individuals showed that Hamiltonella, Arsenopnohus and Fritschea were localized inside the bacteriome, Cardinium and Wolbachia exhibited dual localization patterns inside and outside the bacteriome, and Rickettsia showed strict localization outside the bacteriome. This study is the first survey of whitely populations collected in Brazil, and provides further insights into the complexity of infection with secondary endosymionts in whiteflies.PLoS ONE 09/2014; 9(9):e108363. · 3.53 Impact Factor
Almost There: Transmission Routes of Bacterial
Symbionts between Trophic Levels
Elad Chiel1,2,3*, Einat Zchori-Fein2, Moshe Inbar1, Yuval Gottlieb4, Tetsuya Adachi-Hagimori3,5,
Suzanne E. Kelly3, Mark K. Asplen6, Martha S. Hunter3
1Department of Evolutionary and Environmental Biology, University of Haifa, Haifa, Israel, 2Department of Entomology, Newe-Ya’ar Research Center, ARO, Ramat-Yishai,
Israel, 3Department of Entomology, University of Arizona, Tucson, Arizona, United States of America, 4Department of Entomology, the Volcani Center, ARO, Beit-Dagan,
Israel, 5Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan, 6Department of Entomology, University of Minnesota, St.
Paul, Minnesota, United States of America
Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host’s
reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary
for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but
the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial
symbionts – Rickettsia and Hamiltonella – from their host, the sweet potato whitefly, Bemisia tabaci, to three species of
whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential
for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization
(FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a
Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to
penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically
transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia
proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation.
Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by
feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being
removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested,
and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to
horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in
transmission of species of symbionts among species of hosts are discussed.
Citation: Chiel E, Zchori-Fein E, Inbar M, Gottlieb Y, Adachi-Hagimori T, et al. (2009) Almost There: Transmission Routes of Bacterial Symbionts between Trophic
Levels. PLoS ONE 4(3): e4767. doi:10.1371/journal.pone.0004767
Editor: Jason E. Stajich, University of California, Berkeley, United States of America
Received December 4, 2008; Accepted February 10, 2009; Published March 10, 2009
Copyright: ? 2009 Chiel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the following sources: grant No 2004416 from the United States-Israel Binational Science Foundation (BSF), Jerusalem,
Israel to EZ-F and MSH; The National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant No 2006-35302-17165 to
MSH and EZ-F and The United-States - Israel Binational Agricultural Research and Development fund (BARD), Graduate Student Fellow award No GS-1-2007 to EC.
Contribution No 503/08 from the Agricultural Research Organization, Bet Dagan, Israel. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org.
The occurrence of arthropods serving as hosts for bacterial
symbionts is very common. Primary, obligate symbionts that
provide essential nutrients lacking in the host’s diet, are strictly
maternally transmitted and show congruent phylogenies with
those of their host group [1,2]. Facultative, secondary symbionts
are also transmitted vertically, and promote their own transmission
by contributing to host fitness or by manipulating the host’s
reproduction [3–9]. Phylogenetic trees of secondary symbionts are
largely incongruent with those of their hosts. This, and the fact
that the same secondary symbionts are sometimes found in
distantly related hosts, is attributed to rare horizontal transmission
events of the symbionts between species [1,10,11].
The routes of horizontal transmission are not very well known,
although transmission via common host plants and/or common
natural enemies has been hypothesized, and phylogenetic evidence
for the latter has been provided [12–14]. Rare examples of
experimentally demonstrated natural intra-specific horizontal
transmission include Arsenophonus , Wolbachia  and a virus
 in parasitoids, as well as transmission between mates of the
same aphid species . In contrast, documentation of inter-
specific transmission is almost non-existent. Huigens et al 
showed horizontal transmission of Wolbachia between conspecifics
of Trichogramma kayaki when developing within the same host.
However, attempts to show inter-specific horizontal transmission
of Wolbachia by the same mechanism, between Trichogramma
species, resulted in loss of the symbiont from the recipient species
within a few generations . In lieu of more natural examples,
some microinjection studies have been successful in establishing
some new stable associations [20–23], yet others have been
unsuccessful in establishing novel symbiont-host associations
PLoS ONE | www.plosone.org1 March 2009 | Volume 4 | Issue 3 | e4767
[24–25], suggesting limits to the ability of symbionts to colonize
the germ line of some hosts. While elegant work has shown how
Wolbachia colonizes the germ line of a Drosophila host following
injection of cured individuals , why symbionts fail to become
established is not understood.
The intimate interaction between hosts and their endo-
parasitoids would seem to provide opportunities for horizontal
transmission of symbionts, as parasitoid larvae consume nothing
but symbiont-contaminated food throughout their development.
Yet, to our knowledge, there is no experimental evidence of
permanent acquisition of arthropods’ symbionts by their natural
enemies, hence the notion that inter-specific horizontal transmis-
sion is a rare event.
Here we followed transmission routes of symbionts from their
host – the sweet potato whitefly, Bemisia tabaci – to parasitoids.
Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a minute
insect that feeds on phloem sap of numerous host plants and is a
major pest of agricultural crops . Bemisia tabaci harbors a
primary symbiont, Portiera aleyrodidarum that most probably
produces amino acids lacking in the phloem diet . This
primary symbiont is located only within specialized cells –
bacteriocytes – that are aggregated in two clusters called
bacteriomes . In addition, B. tabaci may harbor a variety of
secondary symbionts: Arsenophonus, Cardinium, Fritschea, Hamiltonella,
Rickettsia and Wolbachia (reviewed in [1,2]; ), whose function is
yet mostly unknown. The B. tabaci colony used in our study carried
only two of those secondary symbionts: Hamiltonella and Rickettsia.
Hamiltonella is located inside the bacteriocytes with the primary
symbiont, while the Rickettsia in our culture is dispersed throughout
the hemocoel .
Bacteria of the genus Rickettsia (a-Proteobacteria) are best known
as vector-borne agents of many vertebrate diseases. The more
recent discoveries of Rickettsia in many different invertebrates, with
diverse effects such as reproductive manipulation, heat tolerance
and plant disease, suggest the disease-causing members represent a
small portion of a much larger group . The Rickettsia in B.
tabaci is most closely related to the pea aphid Rickettsia, is found in
all developmental stages of the whitefly, and is maternally
transmitted . Rickettsia is highly prevalent in B. tabaci
populations , but its benefits to the host, if any, are not clear.
As a matter of fact, Rickettsia was found to inflict some costs on
fitness parameters of B. tabaci [33,34]. Hamiltonella (c-Proteobac-
teria) was described from the pea aphid, Acyrthosiphon pisum, where
it occurs in various tissues both extra- and intra- cellularly and
benefits its host by conferring resistance against parasitoids
Bemisia tabaci is attacked by a wide variety of natural enemies,
including parasitoids of the genera Eretmocerus and Encarsia
(Hymenoptera: Aphelinidae) . These two genera belong to
two different sub-families: Eretmocerus, with 16 species recorded
from B. tabaci , is in the Aphelininae subfamily; Encarsia, with
344 described species, of which 175 species attack whiteflies, is in
the Coccophaginae subfamily . Eretmocerus and Encarsia also
differ markedly in their mode of development: Eretmocerus spp. lay
a single egg under the host venter (i.e., between the host and leaf)
and the first instar penetrates and develops within a vital cellular
capsule inside the host . Encarsia spp., in contrast, lay the egg
directly into the body of their whitefly host .
In preliminary screening we found that two species of
Eretmocerus, Er. eremicus (Rose & Zolnerowich) and Er. sp. nr.
emiratus (Zolnerowich & Rose), were both highly infected with a
Rickettsia that had the same 16S rDNA and citrate synthase gene
sequences as the Rickettsia in their host, B. tabaci. Therefore the
current study was initiated to address two key questions:
1) What (if any) are the routes of transmission of Rickettsia and
Hamiltonella from B. tabaci to the whitefly’s parasitoids?
2)Are symbionts that are acquired by the parasitoids then
vertically transmitted to parasitoid offspring?
Materials and Methods
the study: one that carried Rickettsia (R+) and one that did not (R2).
Rickettsia in these whiteflies was distributed throughout the
hemocoel, the ‘scattered’ phenotype . The presence/absence
of Rickettsia was routinely monitored by diagnostic PCR, as
described below. Additionally, the secondary symbiont Hamiltonella
was established in all individuals of both colonies. Each colony was
reared in a separate room at 2761uC, ca. 60% RH and 16:8 L:D.
Both colonies have been maintained for over two years on cowpea
plants (Vigna unguiculata var. California blackeye).
Eretmocerus sp. nr. emiratus, Er. eremicus and
Encarsia pergandiella were each reared separately on cowpea plants
that were infested with R+B. tabaci nymphs as hosts, inside
transparent ventilated plastic jars. Both sexes of Eretmocerus spp.
pergandiella is an autoparasitoid ; females are primary
parasitoids of whiteflies and males are hyperparasitic, developing
on conspecific or heterospecific immatures. Male En. pergandiella
were thus produced by exposing Er. eremicus larvae and pupae to
adult female En. pergandiella. All parasitoid cultures were kept in a
climate-controlled walk-in chamber (2761uC, ca. 60% RH and
Eretmocerus emiratus and Er. eremicus were fed on
honey containing 50 mg/ml Rifampicin for 48 hrs and were
then released on cowpea plants bearing R2B. tabaci nymphs for
oviposition. This process was repeated for two consecutive
generations. The infection status of the progeny was then
checked with PCR and both species were found to be free of
Rickettsia and Hamiltonella, therefore they were continuously reared
on R2whiteflies under the conditions described above. Encarsia
pergandiella was not treated the same way because neither Rickettsia
nor Hamiltonella were detected in adult wasps after development in
Two B. tabaci (biotype B) colonies were used for
4. PCR analysis.
wasps were ground in a 3 ml droplet of proteinase K solution
(20 mg/ml, Invitrogen). The droplet was then transferred into a
tube containing 50 ml of sterile 10% Chelex beads (Sigma-Aldrich)
in PCR water. The tubes were incubated at 37uC for 1 h, then at
96uC for 8 min and then kept at 220uC until analysis. Two
microliters of the DNA lysate were used as a template for PCR
reactions. The presence of Rickettsia was determined using specific
primers for amplifying 16S rDNA gene fragments: 528F [5-
PCR conditions were: 95uC for 2 min followed by 35 cycles of
92uC, 30 s; 60uC, 30 s; 72uC, 30 s, and final incubation at 72uC
for 5 min. Screening for other B. tabaci symbionts, including
Hamiltonella, was done using the primers and conditions described
in . Reactions were carried in a 10 ml volume containing
4 pmol of each primer, 0.01 mmol dNTP’s, 16 ‘‘Thermopol’’
buffer and 0.4 units of Taq DNA polymerase (New England
Biolabs). PCR products were visualized on 1.5% agarose gel using
To extract DNA, individual whiteflies or
Transmission of Symbionts
PLoS ONE | www.plosone.org2March 2009 | Volume 4 | Issue 3 | e4767
SYBR-Green (Cambrex Bio Science Rockland Inc.). To verify the
identity of the PCR products, bands were eluted, DNA was
purified (QIAquick gel purification kit, Qiagen) and sent for direct
sequencing at the University of Arizona’s sequencing facility. The
resulting sequences were compared to known sequences using the
BLAST algorithm in NCBI. Sequences from whiteflies and
parasitoids were compared to one another using the BLAST 2
Sequence in NCBI.
5. Visualization of Rickettsia using Fluorescence In Situ
FISH of B. tabaci parasitized nymphs,
and adult parasitoids was performed with Rickettsia-specific 16S
rRNA DNA probes, as described in . Stained samples were
whole mountedand viewed
Reproducibility and controls were performed as described in the
above reference (at least 20 individuals of each species). Samples of
Er. eremicus females for TEM were prepared as described by 
Experiments and Experimental design
Hamiltonella in whitefly parasitoids.
on Rickettsia- and Hamiltonella-infected whiteflies were censused for
infection. Using a fine needle, approx. 100 pupae of each wasp
species were removed from leaves and placed in a glass vial with
honey. Samples of the pupae were placed in 96% ethanol for
diagnostic PCR. Newly emerged wasps were transferred to a new
vial with honey and samples were placed in 96% ethanol.
Subsequently, wasps were sampled and placed in ethanol on days
3, 6, 9 and 12 post-eclosion. Infection status was then determined
by diagnostic PCR in 10–13 wasps of each species, at each time
There are three likely routes by which symbionts
can be transmitted from the whitefly host to its parasitoids: 1) the
parasitoid larva acquires symbionts while feeding and developing
in an infected host; 2) the adult female wasps acquire symbionts via
host-feeding (piercing of the whitefly integument with the
Wasps that developed
fromB. tabaci to
Figure 1. A diagram illustrating the design of experiment 7, transmission of symbionts from B. tabaci to parasitoids, and 8, vertical
transmission of symbionts in parasitoids. Infection status is indicated either by red ‘‘+’’ sign or blue ‘‘2’’ sign. R=Rickettsia, H=Hamiltonella.
TRT=treatment. Whitefly hosts are illustrated as small yellow ovals on the (green) leaf disks. To test transmission of symbionts from B. tabaci to
parasitoids, one female parasitoid was introduced to each leaf disk for 24 h, after which they were tested by PCR. From the emerging F1, one or two
females from each replicate were used to continue to the vertical transmission experiment, while the rest of the cohort was tested by PCR (two-five
from each cohort). The emerging F2were all collected and two-five from each cohort were tested by PCR.
Transmission of Symbionts
PLoS ONE | www.plosone.org3 March 2009 | Volume 4 | Issue 3 | e4767
Figure 2. FISH of Er. emiratus stained with Rickettisa specific probe (blue). Left panel-Rickettsia probe fluorescent channel; right panel- overlay
of fluorescent and brightfield channels. Arrows pointing to parasitoid gut. A- parasitoid larva (dark, ovoid sphere in the center of the host). Note
Rickettsia in the parasitoid gut, as well in the whitefly’s body remnants, surrounding the parasitoid. B- parasitoid pre-pupa. C- parasitoid pupae (note
the autofluorescence of the anus and mouthpart); 1C, right image- brightfield channel only. D- parasitoid adult abdomen.
Transmission of Symbionts
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ovipositor followed by consumption of host hemolymph); 3) adult
wasps might acquire the symbiont via feeding on honeydew
secretions of infected whitefly hosts. To test these pathways,
cowpea leaf disks (30 mm diameter) infested with 30–50 R+B. tabaci
nymphs (2ndand 3rdinstars) were placed on 1% agar inside 35 mm
Petri dishes and sealed with screen lids. One male and one female of
R2wasps (cured Er. emiratus and Er. eremicus grown for six
generations on R2whiteflies or R2En. pergandiella directly from
the culture) were introduced onto each leaf disk for 24 hrs and were
then collected to 96% ethanol for PCR analysis. The percentage of
infection status of these adults was used to determine the acquisition
of the symbiont via either host-feeding or feeding on infected
honeydew (scenarios 2 and 3 above). For controls, wasps from the
same sources were introduced onto leaf disks bearing R2whiteflies,
and some wasps were placed directly in ethanol, without exposure to
hosts. The leaf disks bearing parasitized whiteflies were then
incubated for approximately two weeks until wasp progeny
emergence and then two to five (at least one male and one female)
wasps from each disk were collected and placed in 96% ethanol. An
estimate of the percentage of symbiont acquisition via exposure
during development (scenario 1 above) was determined by the
infection status of this second group of wasps. Results were subjected
to a chi-square test (JMP 6.1 software, SAS Institute). Figure 1
illustrates the set up of this experiment, as well as the vertical
transmission experiment (#8, below).
To study whether symbiont acquisition via host feeding was
permanent or transient, another experiment was carried out.
Here, approximately 50 R2wasps were introduced onto a plant
infested with R+B. tabaci nymphs (each species on a separate
plant). After 24 h the wasps were retrieved, half of them were
transferred directly to 96% ethanol and the other half were kept in
glass vials with honey for four days, and then also placed in
ethanol. Twenty wasps of each species were screened for Rickettsia
by PCR: ten wasps from the half that were transferred to ethanol
immediately after the exposure to R+whiteflies, and ten from the
half that were fed on honey after exposure.
8. Vertical transmission experiments.
and Hamiltonella are vertically transmitted between parasitoid
generations, cowpea leaf disks bearing R2B. tabaci nymphs were
prepared and parasitoids of three treatments were randomly
assigned to them: 1) F1adults from the previous transmission
experiment that were exposed to R+hosts during development, i.e.
wasps that have been exposed to the symbionts for one generation
only; 2) wasps that had been reared on R+hosts for many
generations; 3) adults from the horizontal transmission experiment
that emerged from R2hosts (control). One female parasitoid was
To study if Rickettsia
Figure 3. FISH of En. pergandiella stained with Rickettisa specific probe (blue). Left panel-Rickettsia probe fluorescent channel; right panel-
overlay of fluorescent and brightfield channels. A- parasitoid larvae, arrow points to specific signal inside the larva body. B- parasitoid pupa, arrows
pointing to the meconia deposited outside the parasitoid’s body.
Transmission of Symbionts
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introduced onto each leaf disk for 24 hrs and was then placed in
96% ethanol for PCR analysis. The leaf disks were incubated for
approximately two weeks until progeny emergence and then two
to five (at least one male and one female) progeny from each disk
were collected and placed in 96% ethanol for PCR analysis. The
set up of this experiment is illustrated in Fig. 1.
Acquisition and maintenance of Rickettsia and
Hamiltonella in whitefly parasitoids
Almost all pupae of the three studied species carried Rickettsia
and Hamiltonella (Er. emiratus- 11 out of 12 infected; Er. eremicus –
13/13; En. pergandiella females - 10/10; En. pergandiella males- 10/
10). However, infection of adult wasps differed significantly
between the two genera of wasps: Rickettsia did not persist in
adults of the two Encarsia species, while adults of both Eretmocerus
species were virtually all Rickettsia-positive, even 12 days after they
had emerged and fed on honey only (sample size=10 wasps; 9 or
10 tested positive in each sample). In contrast, all Encarsia and
Eretmocerus adults were Hamiltonella-negative (0/10 tested for each
The sequences obtained from the Rickettsia and Hamiltonella
primers were 99% similar to the sequences of ‘‘Rickettsia
endosymbiont of Bemisia tabaci’’ (DQ077707.1) and ‘‘secondary
endosymbiont of Bemisia tabaci 16S ribosomal RNA gene’’
(AY429618.1) respectively. The Rickettsia 16S rRNA sequences
obtained from B. tabaci and parasitoids in this study showed 100%
Localization of Rickettsia
Examination of the symbionts’ localization by means of FISH
shows a concentration of Rickettsia in the center of the Eretmocerus
spp larval body in what seems to be the parasitoid’s digestive tract,
as well as scattered signals outside of the larval body in the
remaining whitefly hemolymph (Fig. 2A). Later on, in the pupal
stage, Rickettsia is aggregated in a kidney (or oval) shape within the
wasp larva, and is more distal, toward the tip of the abdomen
(Fig. 2B). Looking at an image without fluorescence shows an
identical kidney-shaped concentration of small, dark spheres that
are likely meconia (fecal material, typically retained within the
wasp body until late in development) (Fig. 2C). In En. pergandiella,
Rickettsia signals can be seen along the digestive tract of the
Figure 4. Rickettsia (white arrows) in Er. eremicus follicular epithelial cell (FC). The gap between the follicular epithelial cell and the oocyte
(the transition zone - TZ) is due to oocyte resorption. N-nucleus; EnC- endochorion; ExC- Exochorion; VE- Vitellin envelope.
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crescent-shaped third instar larva as well as outside of the larva
(Fig. 3A). In the pupal stage, however, Rickettsia is clearly present
only in the meconia, deposited before pupation on both sides of
the pre-pupal wasp (Fig. 3B). These FISH results are consistent
with the results of the acquisition and sustainability experiment. In
particular, they support the finding that adult En. pergandiella that
developed on R+whiteflies are not infected, and suggest that the
detection of Rickettsia in pupal En. pergandiella by PCR is likely due
to an extraction method that includes the whitefly cuticle and
meconial pellets that surrounds the pupal wasp.
Electron micrographs of Er. eremicus reveal the presence of
bacteria inside the ovaries, within follicular epithelial cells, but not
within the oocytes (Fig. 4). Bacteria were also seen right outside the
ovary, adjacent to the tunica propria, the ovarian envelope (Fig. 5).
The germarium also shows bacteria among stem-, pre-follicle-,
and nurse cell nuclei (Fig. 6). The determination that these
bacteria are Rickettsia is supported by: 1) Denaturating gradient gel
electrophoresis (DGGE) analysis of the bacteria present in Er.
eremicus using general 16S rRNA primers that target most known
bacteria. A single band, corresponding to Rickettsia was found in
this analysis (data not shown). 2) Diagnostic PCR using specific
primers designed for B. tabaci symbionts (Hamiltonella, Wolbachia,
Cardinium, Arsenophonus and Rickettsia) showed bands only for
Rickettsia in the Er. eremicus, as well as for the positive controls in
all other cases (data not shown).
Transmission from B. tabaci to parasitoids
Approximately 30% of the uninfected (R2)
Eretmocerus adult wasps (from both species) that were exposed to R+
whiteflies as adults were subsequently infected with Rickettsia
(Fig. 7A & 7B). The proportion of infected females was
significantly higher than the proportion of infected males (Er.
emiratus: 56% infected females vs. 6.7% infected males, x232=8.8,
P,0.01; Er. eremicus: 44% infected females vs. 11% infected males,
x243=5.8, P=0.016), suggesting that host-feeding, in which
females pierce hosts with their ovipositor and imbibe host
hemolymph, is more likely a source of Rickettsia than feeding on
honeydew (which both sexes do) or simple contact with
contaminated insect surfaces. A much higher proportion of those
wasps that developed inside R+whiteflies were infected: 84% of Er.
emiratus and 93% of Er. eremicus emerged as Rickettsia infected wasps
(Fig. 7A & 7B). Thus, transmission of Rickettsia from infected
whitefly hosts to Eretmocerus occurred at the greatest rate during
parasitoid development, and to a much lower extent via host
feeding by adults. All of the controls, i.e. R2wasps that were not
exposed to any hosts and R2wasps that were exposed to R2hosts,
Figure 5. Rickettsia (bordered) outside Er. eremicus ovary envelope, the tunica propria (TP). FC- follicular epithelial cell; EnC- endochorion;
ExC- Exochorion; VE- Vitellin envelope; Tr- Trachea.
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were Rickettsia-free. Rickettsia infections that were acquired by host
feeding seemed to be largely transient, as the proportion of
infected females decreased sharply four days after removal from
hosts (Er. emiratus: 15/20 infected immediately after exposure to
hosts, vs. 2/10 infected four days later; Er. eremicus: 19/20 and 1/
10 infected at the two time points, respectively). In contrast with
the pattern seen for Rickettsia, Hamiltonella was not detected in any
of the Eretmocerus wasps that fed or developed on Hamiltonella-
infected whiteflies (0/26 tested).
Almost all (15 out of 16) of the adult
females were infected with Rickettsia after exposure to R+hosts,
compared to only one infected male (x232=25.5, P,0.0001)
(Fig. 7C). Rickettsia acquired by host feeding and exposure to
honeydew was also transient in En. pergandiella female adults: 17/20
were infected after exposure to R+hosts, while 0/10 were infected
four days later. None of the wasps that developed inside an R+host
were infected. As was found in Eretmocerus, Hamiltonella was also not
detected in any of the En. pergandiella wasps exposed to infected
whiteflies (0/23 tested).
Eretmocerus wasps that developed inside R2whitefly hosts
emerged as uninfected wasps, even when their mothers were
infected throughout their lifetime (0/20 Er. eremicus, 0/35 Er.
emiratus infected, Fig. 7 A, B). There was no difference between the
two experimental treatment groups, i.e., wasps with multiple
generations of exposure to infected whiteflies prior to the
experiment, and wasps with a single generation of exposure
(parents). These experiments provide no evidence of vertical
transmission of Rickettsia. Vertical transmission was not tested in
En. pergandiella because Rickettsia infection did not persist in the
adults of this species.
Interspecific horizontal transmission of facultative intracellular
symbionts is believed to occur rarely, and little empirical evidence
of such transfers exist [e.g. 20, 21, 23]. That horizontal
transmission between species must have occurred, however, is
amply demonstrated in phylogenetic studies that show little
concordance between host and symbiont phylogenies [1,2]. The
results presented here demonstrate distinct transmission patterns
of secondary symbionts between trophic levels and reveal
differences in those patterns between two closely related parasitoid
host genera. Further, we show that Rickettsia that is ingested during
wasp larval development may penetrate the host hemocoel and
Figure 6. Rickettsia (white arrows) in Er. eremicus germarium area, between nuclei of stem/pre-follicle/nurse cells as well as outside
the ovary, next to the Tunica propria (TP). Note mature oocyte on the bottom left corner area. N-nucleus; EnC- endochorion; ExC- Exochorion;
VE- Vitellin envelope.
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infect the ovaries, but do not appear to invade the developing
oocytes (Figs. 4–6), preventing vertical transmission in the wasp.
The variation we document in the transmission of Rickettsia from
whiteflies to parasitoids highlights two possible views of horizontal
transmission. From an evolutionary point of view (most often used
in the symbiont literature), our results show no transmission of
secondary symbionts from B. tabaci to parasitoids that result in a
heritable infection. From a mechanistic point of view, however, we
document the transmission of a microorganism from one
individual to another, unrelated, individual within the same
generation, a necessary precondition of a novel heritable infection
in a population. Further, we show that symbionts acquired by
feeding may be ultimately excreted (‘‘contamination’’), or invade
the hemocoel and persist throughout the host lifetime, two distinct
and sequential steps in the establishment of a long term
Rickettsia established a transtadial infection in Eretmocerus wasps,
i.e. Rickettsia sustained in Eretmocerus from the larval stage through
adulthood, but was not transmitted vertically. The FISH results
indicate that Rickettsia was concentrated in the lower abdomen of
the adult Eretmocerus wasps (Fig. 2C & 2D). The electron
micrographs show that Rickettsia reached the ovaries of Eretmocerus
but did not penetrate the germ line. Instead, it was found in the
follicular epithelium surrounding the eggs and also in tissues
abutting the ovaries (Figs. 4–6). The fact that Rickettsia is found
within or in close proximity to the ovaries suggests that like other
vertically transmitted bacteria, Rickettsia requires admission to the
germ line for its spread and persistence in host insect populations.
In their thorough study, Frydman et al  found that injected
Wolbachia migrate and enter the Drosophila germline via the somatic
stem cell niche in the germarium, from which follicular epithelial
cells develop. Our results suggest, for Rickettsia at least, that
invading the oocyte may require an adaptation distinct from the
ability to find and invade the ovaries. Nonetheless, the inability of
Rickettsia to invade the germ line of Eretmocerus may be a result of a
defense mechanism of the latter.
The frequency of interspecific horizontal transmission in
endosymbiosis of arthropods is clearly low and variable (excluding
disease agents vectored by ticks etc). Possibly, the paucity of
empirical studies conceals a number of unpublished negative
results. Among published results, the frequency of Wolbachia
horizontal transfer between Trichogramma species sharing a
common host was 0–40% and the vertical transmission within
the recipient species diminished within a few generations .
Similarly, Spiroplasma was horizontally transmitted between two
species of Drosophila by an ectoparasitic mite vector but the
subsequent vertical transmission was very low . Variability of
interspecific transmission success was also demonstrated in the
study of Russell & Moran : pea aphids were injected with three
different symbionts that were obtained from other aphid species.
Two symbionts - Hamiltonella and Arsenophonus - were successfully
established and maintained for multiple generations in their new
host, whereas the third one – Regiella – was not. Grenier et al 
Figure 7. Horizontal transmission (from R+whiteflies to wasps)
and vertical transmission (from R+wasps to progeny) of
Rickettsia to males and females of Er. emiratus (top), Er. eremicus
(middle) and En. pergandiella (bottom). ‘P’ are R2wasps that were
exposed to R+whiteflies for 24 hrs (horizontal transmission via host
feeding and/or honeydew), ‘F1’ are their resulting progeny that
developed in R+hosts (also horizontal transmission), and ‘F2’ are
progeny of F1that were exposed to R2hosts (vertical transmission). The
numbers above the columns are the sample size, n, from which the
proportion of infected wasps was calculated. See also Fig. 1 for this
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PLoS ONE | www.plosone.org9 March 2009 | Volume 4 | Issue 3 | e4767
reported successful horizontal transfer of Wolbachia from one
species of Trichogramma to another via microinjection, followed by
stable vertical transmission, but the efficiency of this process was
low. To the best of our knowledge, the only study that describes
symbiont horizontal transmission from a host to its parasitoid is
that of Heath et al. , in which Wolbachia was weakly
transmitted (3.2%) from an infected Drosophila host to a parasitoid,
and subsequently diminished within four generations. Compared
to these studies, the efficiency of Rickettsia transmission from the
host, B. tabaci, to Eretmocerus wasps was very high and yet no
vertical transmission was observed. Our results therefore support
the notion that invasion of the germ line may be the greatest
challenge for symbionts invading novel hosts. Among parasitoids,
maternally transmitted Rickettsia was so far only found in a leaf
miner parasitoid, where it causes parthenogenesis. However, it is
not known whether this symbiont is present also in its hosts, which
may be indicative of inter-trophic horizontal transmission .
Differences among hosts
Why does Rickettsia establish (even if for only one generation) in
the Eretmocerus adults, whereas it appears to be completely excreted
by the Encarsia? Encarsia embryos and larvae are in intimate, direct
contact with the host’s hemolymph throughout their development,
whereas Eretmocerus become in contact with the host’s hemolymph
only in the third instar, due to the unique capsule in which the
larval wasps reside . Hence, our finding that Eretmocerus
acquire Rickettsia while Encarsia do not is, at first, counterintuitive.
It is possible that these differences relate to the timing of the
deposition of the meconium, fecal material. In En. pergandiella the
mid-gut and the hind-gut are not continuous in early development,
when the larva is in a fluid environment, but join only at the end of
the third instar stage. Subsequently, the prepupal wasp deposits
the meconium, with Rickettsia in it, and then pupates .
Eretmocerus spp., in contrast, excrete the meconium only after the
adult emerges, so meconia, with Rickettsia in them, are present in
the body throughout metamorphosis. It may be that Rickettsia has
the opportunity to invade new tissues during this phase, when
tissues are breaking down and new ones are being built. This idea
is supported by the observation that adult acquisition of the
symbiont by consumption of honeydew or host hemolymph does
not persist. Nevertheless, other routes of infection cannot be
excluded: Rickettsia may get to the ovaries by crossing the larval
mid-gut tissues, which in aphelinid larvae typically bear very few
cells, no typical epithelium and no membranes (Dan Gerling, pers.
Differences between symbionts
Adult wasps of all species in our study acquired Rickettsia but not
Hamiltonella from host feeding. One possible reason for that
difference may be the localization of the two symbionts: Rickettsia is
abundant and accessible in the host hemolymph consumed during
the process of host-feeding while Hamiltonella is sequestered within
the bacteriomes . Another explanation is required, however,
for why Eretmocerus, during their development inside a host, acquire
Rickettsia but not Hamiltonella, since the parasitoid larvae consume
the entire host contents before pupation. Indeed, it seems that
Rickettsia is generally more prone to horizontal transmission (e.g. to
mammalian hosts in the case of the disease agents, or to plants in
the case of insect-vectored plant pathogens) than many facultative
intracellular symbiont lineages. To date, Rickettsia has been found
in many host lineages [2,31], whereas Hamiltonella has so far been
revealed only in aphids, in whiteflies and in one psyllid species
[2,10]. A possible mechanism for a greater propensity for
horizontal transmission is greater symbiont mobility: while little
is known about mobility in most symbiont lineages, some
Rickettsia are able to move between cells and tissues using actin
filaments [46,47]. We know nothing about the mobility of
Hamiltonella, yet the fact that Hamiltonella reside in B. tabaci
bacteriocytes where they are vertically transmitted along with the
primary symbiont Portiera, suggests that Hamiltonella may have
more limited mobility.
Naturally, an interesting question for further research would be
to look for phenotypic effects of Rickettsia infection on the
parasitoids. Preliminary results showed no differences between
R+ and R2 Er. emiratus with regards to fecundity, longevity and
sex ratio, thus more fitness parameters need to be explored to
address this question (Chiel et al., unpublished results).
To conclude, our study is one of few empirical demonstrations
of the routes and barriers to horizontal transmission of facultative
symbionts. These data are especially relevant to the often repeated
idea that parasitoids or predators may be instrumental agents for
moving symbionts from one host lineage to the next. In fact, this
notion has some phylogenetic support [e.g. 12, 48], but in some
cases, enemies have likely been wrongly diagnosed by PCR as
being stably infected when the symbionts are simply present in the
gut along with the prey or host material . Our data suggest
that host-parasitoid transmission may, nonetheless, be one way in
which symbionts acquire new hosts. Given that the symbiont is on
the doorstep of vertical transmission, it is not hard to imagine that
some lineage might, with time, acquire an adaptation that
improves the precision of cell targeting in this new host lineage
to get the symbiont over the threshold. Lastly, our study
underscores how little we currently know about the processes of
dispersal of symbionts to new host lineages, and the within-host
movement and germ-line invasion processes necessary for them to
stay once they get there.
We would like to thank David Bentley for assisting with analyzing the
TEMs, and Ayelet Caspi-Fluger for assisting with the FISH. Technical
assistance was provided by Hyo Kim, Seth Kyselka and Gaelen Burke. We
would also like to thank four anonymous reviewers for their very
constructive comments on the manuscript.
Conceived and designed the experiments: EC EZF TAH MH. Performed
the experiments: EC YG TAH SEK MKA. Analyzed the data: EC EZF
MI YG MH. Contributed reagents/materials/analysis tools: SEK. Wrote
the paper: EC MH. Supervised the research: EZF MI MH. Reviewed the
paper: EZF MI.
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